Radiation is one of the most efficient therapeutic agents for certain types of cancer. However, cytotoxicity induced by ionizing radiation limits the radiation dosage that can be administrated to patients. Alternatively, several DNA modification agents and other adjunct treatments have been utilized as "radiosensitizers" to combine with the standard radiotherapy, thus enhancing the radiosensitivity of the targeted cancer cells. Because of the inherent nonspecificity of current radiosensitizers, the enhancement ratio is relatively moderate, and the applicable range clinically, is limited by side effects. Meanwhile, it has become clear that disruption of DNA repair might dramatically increase radiosensitivity. In this research, we propose to explore the possibility of using DNA double-strand break repair proteins as novel molecular targets for the development of radiosensitizers. Specifically, we will focus on the Ku70 and Ku80 proteins (Ku70/80 heterodimers), which are critical components in the repair of ionizing radiation-induced double-strand breaks in mammalian cells. We plan to identify and validate specific domains of the Ku70/80 heterodimer as novel protein targets for identifying effective radiosensitizers. This study proposes to dissect Ku functional domain using genetic, biochemical, proteolytic digestion/mass spectrometry data, and to express the domains in soluble protein form. Using the expressed recombinant protein domains, we will demonstrate the validity of Ku domains as a molecular target with in vitro biochemical assays, and in vivo dominant negative phenotyping. We also intend to use the combinatorial phage display technique to screen for high-affinity peptides for the interested domains. In viva inhibition studies will be performed by introducing the peptide into cancer cells in a retroviral vector. We will also analyze the structural relationship between the peptide inhibitors and their affected functional domains by NMR. The ultimate goal for this study is to identify the Ku domains that can be used as molecular targets for inhibitory radiosensitizers, and to generate more specific inhibitors/effectors that direct more distinct damages to the cancer cells under inadiation.